The long-term goal of this work is to understand in precise molecular terms how large DNA domains are packaged into an inactive chromatin structure called heterochromatin or silent chromatin. This type of chromatin plays a central role in the regulation of gene expression and maintenance of chromosome stability in organisms ranging from yeast to human. Studies in yeast have identified several gene products that are involved in the assembly of silent chromatin, and the types of complexes that these gene products form have been characterized. We have recently shown that a conserved component of silent chromatin, the Sir2 protein, has a functionally essential ADP-ribosyltransferase activity. This proposal will use purified silencing complexes, together with a chromatin assembly system, to reconstitute silent chromosome domains in vitro. This system will then provide the basis for biochemical experiments to deduce molecular mechanisms and the significance of the enzymatic activity of Sir2 in gene silencing. Silent chromatin is a highly conserved feature of eukaryotic chromosomes. It therefore seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply to other settings. A basic understanding of the molecular events that control chromosome structure and ensure the stable inheritance of cell fate provides not only a frame work for understanding how the process can fail, but also provides substrates and knowledge to design therapeutic strategies based on intervention.